Ferrocene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Ferrocene was assessed to investigate the systemic toxicity and its potential adverse effects on reproduction (including offspring development). The study was performed according to OECD Guideline 422.

Mating performance was unaffected by treatment. An apparent reduction in fertility was observed at the highest dose level with three females showing no implantations. Five additional females at this dose level had implantation sites but no litters; the remaining two pregnant females in this group produced litters but suffered total litter loss post partum. At 5 and 10 mg/kg bw/d, one female with implantations did not produce a litter due to in utero loss and a further female in each group suffered total litter loss post partum.

Pre-implantation loss and post-implantation loss were significantly increased at the highest dose level; no clear effect of treatment was observed at lower dose levels. Pre-implantation loss was slightly but not significantly elevated at 5 and 10 mg/kg bw/d; post-implantation loss was higher at 5 and 10 mg/kg bw/d but without a dose-response relationship. Corpora lutea number at 25 mg/kg bw/d was significantly lower than controls; it is unclear whether this represents a direct effect of the test material or is secondary to pre-implantation loss.

The two dams producing litters at 25 mg/kg bw/d suffered total litter loss. Pup weight gain to Day 4 post partum was slightly lower at 5 and 10 mg/kg bw/d; a dose-response relationship is apparent; however values do not attain statistical significance.

A dosage of 10 mg/kg bw/day was considered to be a No Observed Adverse Effect Level (NOAEL) effects on fertility, with a LOAEL of 25mg/kg bw/day

Short description of key information:
Ferrocene was assessed to investigate the systemic toxicity and its potential adverse effects on reproduction (including offspring development). The study was performed according to OECD Guideline 422.

As effects of treatment were apparent at dosages as low as 5 mg/kg bw/day it was not possible to establish a No Observed Effect Level (NOEL) for systemic toxicity. A dosage of 10 mg/kg bw/day was considered to be a No Observed Adverse Effect Level (NOAEL) effects on fertility, with a LOAEL of 25mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Only study available

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Body wt.range stated 180-200g. Actual-males(269-309g), females(193-221g). The target range for RH (50 ± 20%), achieved range was 39- 86%. Neither deviation from Study Plan was considered to have had no impact on the scientific integrity of the study

Principles of method if other than guideline:

DEVIATIONS FROM STUDY PLAN
The predicted body weight range stated in the Study Plan at the start of treatment was 180-200 g, but this range proved to be too low for the age of animals required. At the start of treatment, males weighed 269 to 309g and females weighed 193 to 221g; all were of the correct age and were considered acceptable for the study. The deviation from Study Plan for the weight ranges for these animals was considered to have had no impact on the scientific integrity of the study.
The Study Plan target range for relative humidity was 50 ± 20% respectively but this was exceeded on a number of occasions during the study (achieved range was 39- 86% RH). While the deviations observed for relative humidity were not ideal, it was considered that they had not impacted on the scientific integrity of the study.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number: 80, (40 males and 40 females)
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 269-309 g; Females: 193-221 g (see Deviations from Study Plan)
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: ad libitu, Pelleted, Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK
- Water: ad libitum, mains drinking water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 2-+3
- Humidity (%): 39-86 - it was considered that this had not impacted on the scientific integrity of the study (see Deviation from Study Plan).
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:

28days

Frequency of treatment:

Daily

Dose / conc.:

5 mg/kg bw/day

Remarks:

Basis: actual ingested

Dose / conc.:

10 mg/kg bw/day

Remarks:

Basis: actual ingested

Dose / conc.:

15 mg/kg bw/day

Remarks:

Basis: actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Several observations were made. Parameters included:
Clinical Observations
Functional Observations-Behavioural Assessments, Functional Performance Tests; Motor Activity.and Forelimb/Hindlimb Grip Strength., Sensory Reactivity
Body Weight
Food Consumption
Water Consumption
Reproduction Screening-Mating, Pregnancy and Parturition, Litter Data, Physical Development
Laboratory Investigations-Haematology, Blood Chemistry
Pathology-Organ Weights, Histopathology

Litter observations:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values generally included all litters reared to termination (Day 5 of age), however at 25 mg/kg bw/day group mean values were calculated for all pregnant animals (where appropriate) and also for those females that littered.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i) Pre-coital Interval
ii) Fertility Indices
Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length
ii) Parturition Index
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values generally included all litters reared to termination (Day 5 of age), however at 25 mg/kg bw/day group mean values were calculated for all pregnant animals (where appropriate) and also for those females that littered.
i) Implantation Losses (%)
ii) Live Birth and Viability Indices
iii) Sex Ratio (% males)

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical signs observed on the study were unremarkable and did not indicate any adverse effect of treatment at 5, 10 or 25 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Both had effects see discussion

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Both had effects see discussion

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

There were no clear effects of treatment on fertility at 5,10 mg/kg bw/day. However, at 5, 10 mg/kg bw/day, 1 female had implantations but was not observed to litter; additionally at both dosages, 1 female showed total litter loss post partum

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, as assessed by pre-coital interval and the evidence of mating observed (copulation plugs, sperm in the vagina), was unaffected by treatment at 5, 10 and 25 mg/kg bw/day.

Dose descriptor:

NOEL

Effect level:

5 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: reproduction and offspring survival, growth and development

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Reproduction and systemic toxicity

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

10 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Dose descriptor:

NOEL

Generation:

F1

Effect level:

5 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: offspring survival, growth and development

Reproductive effects observed:

not specified

Conclusions:

Ferrocene was assessed to investigate the systemic toxicity and its potential adverse effects on reproduction (including offspring development). the study was performed according to OECD Guideline 422.

There were clear treatment-related effects on reproduction at 25 mg/kg bw/day. At 5 and 10 mg/kg bw/day, reproduction per se was not affected by treatment but at both dosage two females failed to maintain their litter to Day 4 of age due to either in utero or post partum offspring losses. Very high pre and post natal losses were evident at 25 mg/kg bw/day, therefore the litter deaths at lower dosages become problematic when establishing a NOEL effect level for offspring survival, which may become clearer in a study of larger dimensions. Within the context of this study, there was no evidence of effects of treatment on surviving offspring at 5 mg/kg bw/day, although there was a suggestion of marginally lower weight gain at 10 mg/kg bw/day.

As effects of treatment were apparent at dosages as low as 5 mg/kg bw/day it was not possible to establish a No Observed Effect Level (NOEL) for systemic toxicity. A dosage of 10 mg/kg bw/day was considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity. In view of this the NOEL for reproduction and offspring survival, growth and development was considered to be at 5 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

One good quality Klimisch score 1 screening study available performed to OECD Guideline 422

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Ferrocene was assessed to investigate the systemic toxicity and its potential adverse effects on reproduction (including offspring development). The study was performed according to OECD Guideline 422.

As effects of treatment were apparent at dosages as low as 5 mg/kg bw/day it was not possible to establish a No Observed Effect Level (NOEL) for systemic toxicity. The study achieved a NOAEL for offspring survival, growth and development of 5 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

no further testing for developmental toxicity is necessary because the substance is known to cause developmental toxicity, meeting the criteria for classification as toxic for reproduction category 1A or 1B: May damage the unborn child (H360D), and the available data are adequate to support a robust risk assessment and the classification and labelling of the substance

Species:

rat

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

One good quality Klimisch score 1 screening study available performed to OECD Guideline 422

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In an OECD 422 study (combined repeat dose toxicity study with reproductive/developmental toxicity screening test), groups of Wistar rats (10/sex) were exposed to Ferrocene (dicylclopentadienyl iron) by gavage (in arachis oil) at dose levels of 0, 5, 10 or 25 mg/kg bw/d for two weeks prior to mating and throughout mating, gestation and early lactation. The study was terminated at Day 4 post partum.

No deaths occurred and no signs of toxicity were observed during the study period. Pre-mating weight gain and food consumption were lower in both sexes. Haematology revealed effects on erythrocyte parameters in both sexes at 25 mg/kg bw/d; lower erythrocyte count was also seen in males at 5 and 10 mg/kg bw/d. Necropsy showed dark and/or enlarged spleens in both sexes at the highest dose level; mild renal effects were also observed in males. Histopathology showed haemosiderin deposition in all treated groups. A NOAEL for systemic toxicity was therefore not established.

Mating performance was unaffected by treatment. An apparent reduction in fertility was observed at the highest dose level with three females showing no implantations. Five additional females at this dose level had implantation sites but no litters; the remaining two pregnant females in this group produced litters but suffered total litter loss post partum. At 5 and 10 mg/kg bw/d, one female with implantations did not produce a litter due to in utero loss and a further female in each group suffered total litter loss post partum.

Pre-implantation loss and post-implantation loss were significantly increased at the highest dose level; no clear effect of treatment was observed at lower dose levels. Pre-implantation loss was slightly but not significantly elevated at 5 and 10 mg/kg bw/d; post-implantation loss was higher at 5 and 10 mg/kg bw/d but without a dose-response relationship. Corpora lutea number at 25 mg/kg bw/d was significantly lower than controls; it is unclear whether this represents a direct effect of the test material or is secondary to pre-implantation loss.

The two dams producing litters at 25 mg/kg bw/d suffered total litter loss. Pup weight gain to Day 4 post partum was slightly lower at 5 and 10 mg/kg bw/d; a dose-response relationship is apparent; however values do not attain statistical significance.

The results of this study demonstrate an effect of treatment on fertility, pre-natal and post-natal survival at a dose level of 25 mg/kg bw/d. Fertility was unaffected at 5 and 10 mg/kg bw/d; it is unclear if effects are male or female-mediated. There is an indication of possible developmental toxicity at 5 and 10 mg/kg bw/d based on slightly elevated pre-implantation and post-implantation loss, total litter loss and reduced offspring weight gain in these groups.

Justification for selection of Effect on developmental toxicity: via oral route:
Only study available

Justification for classification or non-classification

There is clear indication of developmental toxicity in the ferrocene study and there is also evidence for concern on fertility. This concern is considered sufficient enough to trigger classification in Category 1B for reproductive (developmental/ fertility) toxicity (CLP).